During early hematopoietic development, multipotent stem cells proliferate and differentiate into lineage-committed progenitors. This process is currently poorly understood, and it is not known how differentiation and proliferation are regulated by extracellular factors. The ability to influence these events is, however, critical for ex vivo expansion and manipulation of hematopoietic progenitor cells. A better understanding of the early steps of hematopoiesis depends on a more precise definition of progenitor cell types as well as knowledge about molecules that are regulated during the transition from one stage of maturation to the next.
Subsets of hematopoietic progenitor cells respond differently to a large number of cytokines. For certain cytokines, differences in cellular responsiveness have been ascribed to changes in receptor expression during early progenitor cell differentiation. It has recently been demonstrated that the receptor for etythropoietin (EPO-R) is specifically expressed on cells that have undergone commitment to the erythroid lineage. This suggests that the receptor for a lineage-restricted cytokine may serve as an early marker of progenitor cell commitment. M-CSF is another cytokine with highly selective activity. Injection of M-CSF into non-human primates leads to an increase in the number of monocytes in peripheral blood, whereas no changes are observed in the number of other myeloid cells. Colonies formed when bone marrow cells are cultured in the presence of M-CSF alone contain predominantly monocytic cells. When added in combination with other cytokines, M-CSF stimulates the formation of high proliferative potential colonies (HPP-CFCs). This indicates that the ability to respond to M-CSF may be an early event in myeloid development.
The present invention demonstrates that M-CSF responsiveness and the M-CSFR expression can be used to discriminate monocytic and granulocytic cells within a population of cells which strongly expresses the CD34 antigen (CD34.sup.hi). Briefly, the method comprises isolating phenotypically and functionally defined CD34.sup.+ subsets, and staining with anti-M-CSFR monoclonal antibodies to measure expression on these primitive progenitors and cells committed to the granulocytic and monocytic lineages, based upon expression of M-CSFR. CD34.sup.hi M-CSFR.sup.hi cells were highly clonogenic and approximately 70% of the colonies were CFU-M (monocytic), whereas less than 20% were CFU-G (granulocytic). In contrast, CD34.sup.hi cells that were positive for the granulo-monocytic marker CD64 and negative for the M-CSFR contained high frequencies of 91% pure CFU-Gs. After 60 h in culture, CD34.sup.hi M-CSFR.sup.hi cells developed into distinct populations of M-CSFR.sup.hi and M-CSFR.sup.lo cells. These two populations gave rise almost exclusively to monocytes and granulocytes, respectively. This result demonstrates that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M-CSFR and demonstrate that M-CSFR is a useful marker to discriminate CFU-Ms from CFU-Gs.